For successful gene transfer to primitive hematopoietic cells several requirements need to be achieved. These include identification of the desired target cell population, identification of the appropriate vector to be used, and achieving desired levels of gene expression. To date, successful gene transfer in human subjects remain problematic. To address these problems as well as important safety issues, studies in non-human primates are being undertaken to optimize gene transfer to nonhuman primate hematopoietic cells prior to human clinical studies. Vectors that have been evaluated include self-inactivating (SIN) retroviral vectors and adeno-associated viral vectors. These vectors have been constructed to express reporter genes, such as the enhanced green fluorescent protein (EGFP), or therapeutic genes, such as drug resistance genes, erythropoietin. Transduction conditions employed the RGD-containing fibronectin fragment, RetroNectin (CH-296) and a variety of recombinant hematopoietic growth factors, such as stem cell factor (SCF), interleukin-6, megakaryocyte growth and differentiation factor (MGDF or thrombopoietin) and the human Flt-3 (fms-like tyrosine kinase) ligand in either serum containing or serum free media. Viral vectors evaluated include Moloney murine leukemia-based vectors, adeno-associated virus (AAV) vectors, and third generation chimeric human immunodeficiency virus type-1 (HIV-1)-based lentiviral vectors. Third generation lentiviral vectors were pseudotyped with the vesicular stomatitis virus G-protein. Our efforts over the past year have resulted in publications comparing cytokine mobilization therapies to improve hematopoietic stem/progenitor cell mobilization, studies involving novel approaches using AAV vectors to target genetic transfer to the salivary gland, determination of the relative safety of vectors based on insertion site analysis, and the evaluation of novel lentiviral vectors that improve gene transduction of hematopoietic stem cells and contribute to long term hematopoietic recovery of a myeloablated animal. In addition, genetic tracking continues to be performed on cells that have been effectively transduced with a retroviral vector in order to determine their contributions to hematopoietic lineage recovery as well as other tissues such as brain perivascular macrophages. Persistent multiple lineage marking using clinically feasible protocols has been achieved ranging from 5-15%. Efforts are being made to extend this level of marking to stem cells derived from other tissues besides BM and cytokine mobilized PB, such as adult mesenchymal stem/progenitor cells and induced pluripotential stem cells. Phenotypic and functional analyses of cells following hematopoietic reconstitution are being evaluated. Despite these successes, questions remain. How can consistent high levels of expression be obtained using therapeutic genes? Can other stem cells either derived from bone marrow or other easily accessible tissues be targeted to assist in either the contribution or repair of other organs? Future studies are aimed to evaluate therapeutic vectors, improve hematopoietic stem cell recovery and transduction efficiency, further delineate the nature and clonality of populations contributing to the reconstitution using genetic tracking methodologies, and, with the initiation of new studies, to isolate or induce and characterize primitive cell populations which may contribute to organogenesis or the repair of damaged tissues.